1. Field of Invention
This invention relates to a method for the sequential cloning of chromosomal DNA.
2. Background of Related Art
Currently, large sums of money are being spent and great efforts are being made to map the human genome and the genomes of other organisms. Such mapping is of great interest to molecular biologists.
Certain disorders, such as for example, hemophilia and Lou Gehrig's Disease, are associated with defective genetic material. One of the aims of the human genome mapping project is to discover which diseases are associated with defective genetic material. If the structure of such defective genetic material were known, reliable tests could be developed to determine, for example, who would be susceptible to certain forms of cancer, Lou Gehrig's Disease and other genetic disorders.
The circular integration of plasmid DNA into a bacterial chromosome is described by Mejean et al. and by Niaudet et al.; see, Niaudet et al., "Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning" Gene, 19, 277-284 (1982); and Mejean et al , "Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision" Gene, 15, 289-293 (1981). Mejean et al. and Niaudet et al. further disclose that when heterologous DNA in the circularly integrated plasmid have the ability to replicate in a different host bacterium, then a recombinant plasmid may be removed with restriction enzymes, ligated and cloned in that different host bacterium. However, the methods of Mejean et al. and Niaudet et al. are limited to the cloning of specific DNA fragments.
It is a purpose of the invention to provide a method for sequential cloning of genomic DNA.
Another purpose of the present invention is to provide a method for the sequential mapping of a genetic sequence.
Still a further purpose of the present invention is to provide a method to be used in conjunction with the mapping of the human genome.
A method for sequentially synthesizing gene length segments of DNA is described in U.S. Pat. No. 4,293,652. The method includes synthesizing a fragment of double stranded DNA corresponding to a preselected portion of the length of DNA to be synthesized or "cloned". A fragment of the preselected DNA is inserted into the cloning vector. The vector is cloned in an appropriate host. Next, the vector is opened by a restriction enzyme which cuts at the end of the first fragment. Another segment of the preselected DNA is then inserted into the vector. The process is repeated until the entire length of the preselected DNA has been inserted into the vector. The entire length of the preselected DNA may then be cloned by inserting the vector into a suitable host. This method sequentially synthesizes and clones an already known segment of DNA, unlike the method of the present invention, which sequentially clones and maps an unknown segment of DNA.
For a better understanding of the present invention reference is made to the following description, taken together with the accompanying drawings, and its scope will be pointed out in the appended claims.